Læknablaðið : fylgirit - 01.09.1993, Blaðsíða 37

LÆKNABLAÐIÐ/FYLGIRIT 24
35
CLONING AND CHARACTERIZATION OF
THE HUMAN AND MOUSE HOMOLOGS OF A
SODIUM DEPENDENT PHOSPHATE
TRANSPORTER
Kristleifur Kristjánsson, Samuel S. Chong, Christine
A Kozak, James E. Bourdeau and Mark R. Hughes.
Institute for Molecular Genetics, Baylor College of
Medicine, Houston, Texas.
X-linked hypophosphatemic rickets (XLH) is the most
commonly inherited bone disease in man. In addidon to
bowing of weight bearing bones with secondary pain and
restricted movements of joints, this condition is
characterized by phosphaturia in the presence of lowered
serum phosphate. The primary defect has long been
assigned to a defective sodium dependent phosphate
transponer in the proximal convoluted tubule of the
kidney. Cloning of the gene for this transporter would be
instrumental in understanding the pathophysiology of the
disease. We have cloned and sequenced the murine and
human homologs of a sodium dependent phosphate
transport gene. The open reading frames are 1393 and
1400 base pairs for the mouse and human cDNA
respectively, producing predicted protein products with
molecular weights of approximately 51,500. There is
approximately 75% homology at the nucleotide level
between the mouse and the human cDNA. We have
mapped the genes to chromosome 13 in the mouse and
chromosome 6 (p21.3-p23) in the human.
Northem blots using RNA from various mouse
tissues show the expression to be kidney specific. Using
antisense mRNA to the mouse clone as a probe, in situ
hybridization against mouse renal tissue sections shows
significant localization of the transcript to cells of the
proximal convoluted tubules compared to other cells of the
kidney. The autosomal localization of these genes
excludes them as candidate genes for XLH and the mouse
model to the human disease, Hyp. Functional analyses
and characterization of these gene products will help to
define theirrole in phosphate transport and mineral
homeostasis.
PREIMPLANTATION PREVENTION OF X-
LINKED DISEASE: RELIABLE AND RAPID
SEX DETERMINATION OF SINGLE HUMAN
CELLS BY RESTRICTION ANALYSES OF
SIMULTANEOUSLY AMPLIFIED ZFX AND
ZFY SEQUENCES
Kristleifur Kristjánsson, Samuel S Chong, Juan
Cota and Mark R. Hughes. Institute for Molecular
Genetics, Baylor College of Medicine, Houston, Texas.
We present a reliable PCR-based strategy for sex
determination at the single cell level using a restriction
enzyme polymorphism to distinguish between the co-
amplified region of ZFX and ZFY. A nested set of
primers was designed to simultaneously amplify a 344bp
exon segment of the zinc finger domain of ZFX and ZFY
which lie centromeric to the pseudoautosomal region of the
respective sex chromosomes. Digestion of the amplified
ZFX segment with HAEIII yields two firagments of 44bp
and 300bp, in contrast to three fragments of 44bp, 84bp
and 216bp when the ZFY segment is digested. Therefore,
an XY genotype can be clearly distinguished on a
polyacrylamide minigel by the presence of 4 bands (44,
84, 216, 300) as opposed to 2 bands (44, 300) for an XX
genotype. The entire analysis is routinely performed in
less than 6 hours. We tested the efficacy of this strategy in
a blinded analysis of single human lymphoblasts,
amniocytes and chorionic villus cells that included blanks
as negative controls. Cells were individually
micromanipulated into separate PCR reaction mbes under
conditions that avoid exogenous DNA/cell contamination.
Of a total 206 amplification tubes 173 of 174 (99.4 %
V 9
sensitivity) containing single cells successfully amplified,
and none of the blank mbes (0/32) showed evidence of
product All 173 of the single cells that amplified were
correctly typed as XX or XY (100% assay specificity).
This strategy avoids a source of misdiagnosis obtained in
methods where Y-chromosome repeat sequences are used
but fail to amplify in XY cells, incorrectiy resulting in an
XX diagnosis. It also represents a simplification over
methods which amplify separate sequences on the X and Y
chromosome, since ZFX &ZFY can be amplified with the
same primer pair. The presence of a common HaelII site
in both X and Y derived fragments serves as a built-in
control for failure of enzyme digestion. This strategy is
useful for rapid determination of the genotypic sex in
newboms with ambiguous phenotype, and in forensic
analysis where small samples are common. In
preimplantation diagnosis, this method offers reliable
single blastomere analysis of the human cleavage stage
embryo to avoid uncharacterized, lethal X-linked genetic
disease prior to pregnancy.